mCherry-EB3 was imaged using the 561 nm laser at 2 s per frame for 5 min. For selleck chemicals llc LAMP1-RFP photobleaching experiments, images were acquired using the 561 nm laser at 2 frames per second for 5 s prior to and for 120 s subsequent to photobleaching. For FRAP, images were acquired using the 488 nm laser at 1 frame per second for 5 s prior to and for 180 s subsequent to photobleaching. FRAP curves were fit to the single exponential equation, f(t) = A(1 − e−τt), where A is mobile fraction. Kymographs from time-lapse imaging data were made

using the Multiple Kymograph plugin (submitted by J. Rietdorf and A. Seitz, European Molecular Biology Laboratory, Heidelberg, Germany) in ImageJ (NIH). All tracks from each LAMP1-RFP kymograph were classified as either anterograde, retrograde,

or nonmotile. Cargos that moved net distances greater than 10 μm in a single direction were classified as either anterograde or retrograde. Cargos that moved less than 10 μm were classified as nonmotile. The tracks of individual lysosomes, moving greater than 10 μm, were determined in ImageJ by marking points where the slope changes and interpolating the intermediate points of the track using a linear function. The instantaneous velocities, pauses per track and motility switches per track were calculated from this data. A pause was defined as an instantaneous velocity less than 100 nm per second. A motility switch is defined as a change in the unless direction of motility (anterograde to retrograde or vice versa) or switch from directional motility (anterograde or retrograde) to a pause and Bcl 2 inhibitor vice versa. Line-scan fluorescence intensity quantification was performed on raw imaging data using Metamorph. A line starting at the distal end of the neurite was drawn along the process toward the cell

body and the florescence intensity was measured along the line. The fluorescence intensity of both channels was normalized to the minimum value of each line. The normalized intensities were divided by the corresponding normalized GFP intensity and plotted as a function of distance from the neurite tip. The retrograde flux of LAMP1-RFP was measured from kymographs of the photobleached region of the axon, which were made prior to and subsequent to the photobleaching in Volocity (PerkinElmer). Vesicles originating from the distal end and moved at least 50 pixels (3.5 μm) into the photobleached zone were considered retrograde moving cargos. To test for coimmunoprecipitation of dynactin, COS-7 cells were cotransfected with Myc-tagged p150Glued isoforms and HA-tagged wild-type p150Glued using FuGENE 6 (Roche) for 24 hr. Cells were lysed in 100 mM PIPES, 1 mM EGTA, 2 mM MgCl2, 25 mM NaCl, 0.5 mM DTT, 1% Triton X-100, and protease inhibitors (1 mM PMSF, 1 mM Leupeptin, 0.001 mg/ml Pepstatin-A and 0.01 mg/ml TAME).